3-mt antigens, 3-mt recombinant monoclonal antibodies and uses thereof

Abstract

The present application relates to the technical field of biology, and in particular to a 3-MT antigen, a 3-MT recombinant monoclonal antibody and application thereof. The present application adopts 3-MT to couple KLH through an intermediate arm, so as to obtain an activated form of 3-methoxy dopamine antigen. The antigen is used to immunize rabbits, a phage library is constructed, and a 4354# antibody is obtained through panning and screening. Experiments show that the antibody has high titer, good gradient, and meets the requirements of the antibody of a magnetic microparticle chemiluminescence 3-MT kit in terms of specificity, repeatability and stability, and fills the market gap.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to 3-MT antigen, 3-MT recombinant monoclonal antibody and applications thereof. Background Art

[0002] Catecholamines are neurotransmitters containing catechol and amine groups. These groups are enzymatically linked to L-tyrosine in the sympathetic nerves, adrenal medulla, and chromotropic cells. Commonly, catecholamines refer to dopamine, norepinephrine, and epinephrine. These three catecholamines are derived from tyrosine as a precursor. Three metanephrine metabolites include 3-metanephrine (MN), 3-methoxynorepinephrine (NMN), and 3-methoxydopamine (3-MT), which are 3-oxymethylated metabolites of epinephrine, norepinephrine, and dopamine, respectively.

[0003] Pheochromocytomas and paragangliomas (PPGLs) are a group of neuroendocrine tumors originating from the adrenal medulla or extraadrenal sympathetic nerve chain. They are one of the causes of secondary hypertension, with a prevalence of 0.2% to 0.6% in general hypertension clinics.

[0004] Most PPGLs can occur in all age groups, with the peak incidence between 30 and 50 years old, and an annual incidence of 2 to 8 per million people. PPGLs usually secrete catecholamines (CA) in a paroxysmal or persistent manner, with typical clinical manifestations of paroxysmal or persistent hypertension and a triad of headache, palpitations, and sweating. Excessive release of CA can induce severe cardiovascular manifestations, including Takotsubo cardiomyopathy, hypertensive crisis, and acute myocardial infarction, ultimately developing into multi-system failure and death. Therefore, timely diagnosis is particularly important.

[0005] 3-MT is an intermediate metabolite of catecholamines. Its metabolic pathway involves the conversion of dopamine to 3-methoxytyramine (3-MT) via the enzyme COMT. Early studies have shown that plasma 3-MT can be used to detect dopamine (DA)-secreting polyneuromas (PPGLs) or head and neck paragangliomas (HNPGLs). Furthermore, 3-MT is considered a novel biomarker for metastatic PPGLs. Studies have shown that plasma 3-MT levels are significantly elevated in patients with metastatic PPGLs compared with those without metastasis. Receiver operating characteristic (ROC) curve analysis indicates that plasma 3-MT levels are more accurate in predicting metastatic PPGLs than methylnorephinephrine. High plasma 3-MT levels are associated with poor disease-specific survival (DSS) in patients with metastatic PPGLs and / or HNPGLs, suggesting that 3-MT may play a role in the personalized management and follow-up of patients with metastatic PPGLs and / or HNPGLs. The "Expert Consensus on the Diagnosis and Treatment of Pheochromocytomas and Paragangliomas 2020" recommends 3-MT as a biochemical marker for PPGL detection.

[0006] In summary, hypertension is a common chronic disease. PPGL, a major cause of secondary hypertension, can lead to uncontrollable malignant hypertension and cardiovascular and cerebrovascular diseases. Therefore, early detection and timely diagnosis of PPGL are of great significance. 3-MT provides accurate judgment for the diagnosis of PPGL and the differentiation of benign and malignant types.

[0007] Currently, there are no immunoassay products available for 3-MT detection in China or abroad. High-performance liquid chromatography (HPLC) is the primary method, but HPLC testing is time-consuming, costly, and widely varying laboratory conditions make it difficult to achieve mass production. Therefore, screening for 3-MT antibodies for application in magnetic microparticle luminescence platforms and other related catecholamine projects has become a critical need.

[0008] Dopamine analogs such as 3-MT, NMN, and MN are all small molecules with very similar structures. Therefore, developing antibodies and detection kits with good gradients and high specificity is extremely difficult. Providing a 3-MT neoantigen for screening for highly specific and potent antibodies, and subsequently developing a 3-MT detection kit with excellent specificity, high accuracy, and high sensitivity, is of great practical significance. Summary of the Invention

[0009] In view of this, the present invention provides 3-MT antigen, 3-MT recombinant monoclonal antibody and applications thereof.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] 3-methoxydopamine hapten, the structural formula of which is shown in Formula I:

[0012]

[0013] Formula I;

[0014] Wherein, n is any integer selected from 0 to 8.

[0015] In some embodiments, n is equal to 6, and the structure of the 3-methoxydopamine hapten is as shown in Formula II:

[0016]

[0017] Formula II.

[0018] The present invention also provides a 3-methoxydopamine complete antigen, which is a conjugate of the 3-methoxydopamine hapten and a carrier protein;

[0019] The carrier protein includes one or more of KLH, bovine serum albumin, human serum albumin, ovalbumin, etc.

[0020] In the present invention, the complete 3-methoxydopamine antigen is a conjugate obtained by coupling the amino group at the terminal of the 3-methoxydopamine hapten with the amino group of a carrier protein.

[0021] In some specific embodiments, the 3-methoxydopamine complete antigen, referred to as 3-MT-BS3-KLH, has a structure as shown in Formula III:

[0022]

[0023] Formula III.

[0024] The present invention also provides a method for preparing the complete 3-methoxydopamine antigen, comprising the following steps:

[0025] Synthesize a hapten with the structure of formula I;

[0026] The hapten is coupled to the carrier protein, and the coupled product is dialyzed to obtain the complete 3-methoxydopamine antigen.

[0027] In some embodiments, the coupling reaction conditions are: shaking reaction at 18-27° C. overnight, and the shaking reaction time is ≥16 h.

[0028] The present invention also provides the use of the hapten or the complete antigen in any of the following:

[0029] (1) Preparation of 3-MT recombinant monoclonal antibody;

[0030] (2) Preparation of products for detecting the levels of 3-MT and its derivatives;

[0031] (3) Preparation of diagnostic products for pheochromocytoma and / or paraganglioma.

[0032] The present invention provides a 3-MT recombinant monoclonal antibody, which is a 3-MT recombinant monoclonal antibody prepared by immunizing rabbits with the 3-methoxydopamine complete antigen.

[0033] The present invention provides a 3-MT recombinant monoclonal antibody, wherein the three CDR regions of the heavy chain respectively have:

[0034] A), the amino acid sequence shown in SEQ ID NO: 1 to 3; or

[0035] B) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in A); or

[0036] C) has at least 80% of the sequence shown in A) or B).

[0037] The three CDR regions of the light chain of the antibody respectively have:

[0038] D), the amino acid sequence shown in SEQ ID NO: 4 to 6; or

[0039] E) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in D); or

[0040] F) and the sequence shown in D) or E) have at least 80% of the sequence.

[0041] In some embodiments, the 3-MT antibody of the present invention comprises a heavy chain variable region and a light chain variable region;

[0042] The light chain variable region has:

[0043] G), the amino acid sequence shown in SEQ ID NO: 7; or

[0044] H) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in G); or

[0045] I), and the sequence shown in H) or I) have at least 80% of the sequence.

[0046] The heavy chain variable region has:

[0047] J), the amino acid sequence shown in SEQ ID NO: 8; or

[0048] K) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in J); or

[0049] L), and the sequence shown in J) or K) have at least 80% of the sequence.

[0050] In some embodiments, the constant region of the heavy chain is of IgG type; the constant region of the light chain is of K type.

[0051] In some specific examples, the present invention utilized the aforementioned 3-MT-BS3-KLH antigen screening to obtain the 3-MT recombinant rabbit monoclonal antibody described herein, designated 4354#. Experimental results demonstrated that this monoclonal antibody exhibited strong specificity, good reproducibility, and high sensitivity, all superior to the control antibody 1605#.

[0052] The present invention also provides a method for preparing the 3-MT recombinant rabbit monoclonal antibody, comprising: immunizing the spleen of a rabbit with the 3-MT-BS3-KLH antigen, constructing a phage library, and obtaining the anti-TPO mouse monoclonal antibody through panning and screening.

[0053] The present invention also provides a biomaterial, characterized in that it comprises any one of the following:

[0054] (I) a nucleic acid encoding the 3-MT antibody;

[0055] (II) an expression cassette or recombinant vector comprising the nucleic acid described in (I);

[0056] (III) A host comprising the nucleic acid of (I) or the recombinant vector of (II).

[0057] The present invention also provides the use of any one of the following a) to b) in preparing a detection product:

[0058] a) the 3-MT recombinant monoclonal antibody of the present invention;

[0059] b) the biomaterial according to the present invention;

[0060] The detection products include: products for detecting the level of 6-carbonylated derivatives of 3-MT, or diagnostic products for pheochromocytoma and / or paraganglioma.

[0061] In the application of the present invention, it is characterized in that the 3-MT derivative is a compound of the following formula IV:

[0062]

[0063] Formula IV.

[0064] The present invention also provides a kit for detecting the level of 3-MT derivatives, which comprises the complete antigen and / or the 3-MT recombinant monoclonal antibody of the present invention.

[0065] In some embodiments, the kit is a magnetic microparticle chemiluminescence kit, comprising magnetic microparticles coated with the antibody of the present invention. Furthermore, the magnetic microparticle chemiluminescence kit also includes a labeled antigen. The antigen in the labeled antigen is the compound represented by Formula IV above. The kit also includes other commonly used components, such as buffers, wash solutions, and blocking solutions. The present invention does not specifically limit the specific types of these components; any commonly used components in the art may be used.

[0066] The present invention also provides a method for detecting the level of 3-MT or its derivatives for non-diagnostic purposes, comprising: detecting a sample to be tested using the kit of the present invention. In some specific embodiments of the present invention, the sample to be tested is a 6C-acylated serum sample.

[0067] In the present invention, a plasma acylating agent is used to treat a test sample to obtain a 6C-acylated serum sample. The plasma acylating agent includes a reagent capable of converting 3-MT into a 3-MT derivative represented by Formula IV. In a specific embodiment of the present invention, the structure of the acylating agent and the acylation reaction process are as follows:

[0068] +

[0069] To achieve specific detection of 3-MT, the applicant, after years of research, attempted to screen antibodies using 3-MT antigens of varying structures. Ultimately, they obtained the highly titered and well-graded 4354# antibody using a complete antigen prepared from the hapten represented by Formula II. Antibodies prepared from complete antigens other than those in Formula I and other antigens outside of Formula I exhibited suboptimal titers and / or gradients, significantly inferior to the 4354# antibody. Furthermore, the applicant also performed various pretreatments on serum samples (including 12C acylation, the 6C acylation described herein, and PEGylation). Results showed that testing serum samples after 6C acylation significantly improved detection sensitivity and specificity, offering distinct advantages for 3-MT detection.

[0070] The present invention utilizes 3-MT coupled to KLH via an intermediate arm to obtain an activated form of 3-methoxydopamine antigen. This antigen was used to immunize rabbits, construct a phage library, and screen the resulting 4354# antibody. Experimental results indicate that this antibody exhibits high titer, a good gradient, and specificity, reproducibility, and stability that meet the requirements of magnetic microparticle chemiluminescent 3-MT kits, filling a market gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The electrophoresis results of 3-MT4354# antibody are shown, the heavy chain is 53±3KD, and the light chain is 22±3KD;

[0072] Figure 2 HPLC results of 3-MT4354# antibody. DETAILED DESCRIPTION

[0073] The present application provides 3-MT antigen, 3-MT recombinant monoclonal antibody and application thereof. Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0074] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0075] The present application couples KLH through a connecting arm BS3 at a specific position of 3-methoxydopamine to obtain a new antigen 3-MT-BS3-KLH with immunogenicity.

[0076] The present application uses the immunogen 3-MT-BS3-KLH to immunize rabbits, constructs a phage library, and then washes the antigen 3-MT-8C-BSA, increases 3-MT-6C competitive washing, and uses 3MT monoclonal screening strategy: preliminary screening of better clones: only using 2-point evaluation (very low, very high), using 0 pg / ml value and 1500 pg / ml calibrator; simultaneous screening of main cross materials: main cross material NMN 2-point detection (low point, high point), 150 and 15000 pg / ml detection, screening cross rate low clone retest advantage clone: select calibrator and cross rate better clone retest, increase calibrator gradient: 0, 40, 200, 1000 pg / ml, select 0 / 40 point better clone; increase cross material screening: NMN / MN repeated determination, determine the cross rate is normal; finally screen out 1 strain of 3-MT antibody, named 3-MT8574#. The experiment shows that it meets the needs of the kit.

[0077] The light chain variable region of the 3-MT8574# antibody has the amino acid sequence shown in SEQ ID NO: 7, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8.

[0078] The sequence of the light chain variable region is SEQ ID NO. 7 as follows:

[0079] ALVMTQTPASVSVPVGDTVTIDC QASESVYNNNHLS WFQQKPGQPPKQLIY GASTLDSGVSSRFKGSGSGTQFTLTITDVVCDDAATYYC AGYKSLTSDGRA FAGGTEVVVK.

[0080] The light chain variable region includes three CDR regions, which are as follows:

[0081] CDR1: QASESVYNNNHLS (SEQ ID NO.1);

[0082] CDR2:GASTLDS (SEQ ID NO.2);

[0083] CDR3: AGYKSLTSDGRA (SEQ ID NO. 3).

[0084] The sequence of the heavy chain variable region SEQ ID NO.8 is as follows:

[0085] QEQLKESGGRLVTPGTPLTLTCTASGFSLS NYYMS WVRQAPGKGLEYIG TIYGVGNTFYASWAKG RFTISKTATTVDLKVTSPTTEDTATYFCGR DAPSVGADL WGPGTLVTVSS.

[0086] The heavy chain variable region includes three CDR regions, which are as follows:

[0087] CDR1: NYYMS (SEQ ID NO. 4);

[0088] CDR2:TIYGVGNTFYASWAKG(SEQ ID NO.5);

[0089] CDR3: DAPSVGADL (SEQ ID NO. 6).

[0090] In the present invention, the 3-MT8574# antibody further comprises a heavy chain constant region and a light chain constant region. Furthermore, the heavy chain constant region is of IgG type; the light chain constant region is of Kappa type. In some specific embodiments, the sequence of the heavy chain constant region is as shown in SEQ ID NO. 9:

[0091] GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK。

[0092] 轻链恒定区的序列如SEQ ID NO.10所示:

[0093] RDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC。

[0094] Pcombxsss 283质粒的序列如SEQ ID NO.11所示:

[0095]

[0096] The present invention will be further described below in conjunction with the embodiments:

[0097] Example 1 Preparation of 3-MT-BS3-KLH Antigen and Antibody

[0098] 1. Synthesis of Antigens:

[0099] The reaction process includes the following steps:

[0100] 1.1 Cross-linking: Slowly add 0.932 mg (186 μL) of dissolved 3-MT antigen dropwise to 20 mg (2 mL) of KLH solution, shaking the mixture to ensure thorough mixing. Slowly add 2.288 mg (114 μL) of BS3 dropwise to the 3-MT and KLH mixture, shaking the mixture to ensure thorough mixing. After mixing, shake the mixture overnight at room temperature (room temperature: 18-27°C, reaction time ≥ 16 hours).

[0101] 1.2. Dialysis: Prepare a dialysis bag (14kd, MD25), cut the bag, and place it in purified water. Let it sit for 0.5 hours, then replace the purified water. Dialyze the sample four times with 0.01M PBS pH 7.2 (2 L per dialysis). Change the solution three times during the first dialysis session. The first dialysis session lasts 3 hours, the second dialysis session lasts 3 hours, the third dialysis session lasts 3 hours, and the fourth dialysis session lasts overnight for 15-16 hours.

[0102] 1.3. Sample Preparation: Undry the dialysis bag, measure the volume with a 5ml pipette, and transfer to a 10ml brown glass vial. Measure the concentration using an A280, label, and store at -20°C.

[0103] 2. Rabbit immunization

[0104] The 3-MT-BS3-KLH antigen was diluted with PBS (0.01M, pH 7.4) and fully emulsified with Freund's adjuvant at a 1:1 volume ratio. The immunization dose was 1 mg / rabbit for the first immunization and 0.5 mg / rabbit for the second immunization. Immunization was administered by subcutaneous injection at multiple sites throughout the body. The first and second immunizations were separated by 30 days, and the second and third immunizations were separated by 30 days. The maximum immunization dose per rabbit was 0.8 ml.

[0105] The titer and gradient were determined by plate chemiluminescence assay; when the titer was 128K and the IC50 reached 400 pg, a rabbit phage library was constructed.

[0106] 3. Nucleic Acid Extraction and Library Preparation

[0107] RNA was extracted from rabbit spleen, and cDNA was reverse transcribed. The variable regions of the light and heavy chains were amplified by PCR, and then ligated with the vector pCOMb3xss (sequence shown in SEQ ID NO. 11).

[0108] 3.1 PCR reaction system (50 μl system):

[0109] VL (light chain variable region) mix: 50ng;

[0110] VH (heavy chain variable region) mix: 50ng;

[0111] 2933 (10 μM): 1.5 μl;

[0112] 2934 (10 μM): 1.5 μl;

[0113] 2× Primerstar Max: 25 μl;

[0114] High-pressure purified water: make up to 50 μl system.

[0115] PCR reaction parameter settings:

[0116] 98℃, 5min;

[0117] 95℃, 15s, 56℃, 15s, 72℃, 25s, 30cycles

[0118] 72℃, 10min.

[0119] 3.2 Enzyme digestion reaction system (50 μl system, SCFV: 50 μl / tube * 5 tubes; pcomb3xss: 50 μl / tube * 8 tubes):

[0120] scFv: 2 μg;

[0121] sfiI (5u / μl): 2.5μl;

[0122] 10× buffer G: 5 μl;

[0123] High-pressure purified water: make up to 50 μl.

[0124] Pcomb3xss: 4 μg;

[0125] sfiI (5u / μl): 2.5μl;

[0126] 10× buffer G: 5 μl;

[0127] High-pressure purified water: make up to 50 μl.

[0128] 3.3 Ligation reaction:

[0129] Use a 20μl system: vector: 0.4μg scFv: 246ng ligase: 1μl 5× ligase buffer: 10μl high-pressure purified water (make up to 50μl). Ligation parameters: incubate at 37°C overnight (14-18 hours). Transform E. coli TG1 cells with high efficiency by electroporation. Count colonies on the plate the next day.

[0130] 4. Calculation of storage capacity and electrical conversion efficiency:

[0131] Reservoir capacity after electroporation = colony count * dilution gradient / coating volume * 30ml, unit is cfu;

[0132] Electroporation efficiency = colony count * dilution gradient / coating volume * 30 ml / input nucleic acid amount (μg) cfu / μg.

[0133] Electroporation efficiency ≥ 10^9 cfu / μg. Gene library capacity ≥ 10^8 cfu. This should meet experimental requirements; otherwise, the gene library needs to be reconstructed.

[0134] 5. Preparation of Phage Antibody Library

[0135] 5.1 Add 5 x 1012 - 4 x 1013 pfu of helper phage M13KO7 (1:20 ratio) to the electroporated library and incubate at 37°C for 30 minutes. Then, incubate at 37°C, 250 rpm, for 1 hour. (The helper phage should be vortexed before use.) Centrifuge at 4500 rpm for 10 minutes, discard the supernatant, add 120 ml of AK medium, and incubate at 250 rpm, 30°C, overnight.

[0136] 6. Titer Determination

[0137] 6.1 Centrifuge the overnight culture at 8000 rpm for 10 minutes and collect the supernatant. Transfer 40 ml of the supernatant to three 50 ml conical centrifuge tubes containing 10 ml of 5xPEG8000 / NaCl. Mix thoroughly and place on ice or in a 4°C refrigerator for 20 minutes. Centrifuge at 8000 rpm for 10 minutes at 4°C and discard the supernatant. Centrifuge at 8000 rpm for 2 minutes at 4°C and remove all excess liquid.

[0138] Add 2 ml of 1x PBS (Gibco) to each tube to resuspend the pellet. Centrifuge at 8000 rpm for 5 minutes. Transfer the supernatant (phage antibody library) to a new centrifuge tube. Add 6% DMSO, mix thoroughly, and aliquot into 1.5 ml centrifuge tubes. Store at -20°C.

[0139] 6.2 Take 20 μl of phage and 180 μl of 2YT and make 10-fold serial dilutions: 101 -10 10 Then add 180 μl TG1 (OD600 = 0.4-0.6) (prepared in 3.5.2.1) and incubate at 37℃ for 30 min. 9 -10 10 Spread 100 μl of the bacterial solution onto Amp+2YT plates. After the plates dry, invert and incubate them at 37°C overnight. Statistical data is collected the next day. The phage titer is also recorded. The statistical formula is: Titer = 2*Ax*10 x+1 pfu / ml; (X: dilution factor, Ax: number of clones at X-fold dilution ratio).

[0140] 7. Main steps of antibody screening

[0141] The day before, coat immunotubes with 1 ml of the immunogenic antigen 3-MT-BS3-BSA (20 μg; 10 μg in the second round; 5 μg in the third round) according to the panning protocol. The next day, discard the coating solution, wash the immunotubes three times with 0.05% PBST, add 5 ml of casein, and block at 37°C for 1 hour. Transfer 1 ml of centrifuged phage to the immunotubes and incubate at 37°C, 70 rpm, for 2 hours. Discard unbound phage from the immunotubes and add 4 ml of 0.05% PBST wash buffer to each tube. Wash the tubes (6 times in round 1, 8 times in round 2, and 10 times in round 3, increasing in sequence), tapping gently for 30 seconds between washes. Wash twice with 1x PBS. Add 1 ml of 3-MT-6C elution buffer (5 μg in round 1, 10 μg in round 2, and 20 μg in round 3) to each tube and incubate at 37°C, 70 rpm, for 60 minutes. The dissociated phages were transferred to a new 1.5 ml centrifuge tube.

[0142] Quickly add 1 ml of eluted phage to 5 ml of TG1. Add 1 ml of TG1 to the corresponding immunotube and incubate at 37°C for 30 minutes. (Mix the bacteria in the immunotube and centrifuge tube, mix thoroughly, and dilute 10 μl to measure the titer (10 μl of the bacterial solution is diluted 10-fold, 101-105: 20 μl phage + 180 μl 2YT). Spread 100 μl to calculate the stock volume.) Add 6 μl of A+ and incubate at 37°C, 250 rpm, for 1 hour. Add M13K07 (MOI = 5) and infect at 37°C for 30 minutes. Add 10 ml of 2% AG-2YT and incubate at 37°C, 250 rpm, for 1 hour. Centrifuge at room temperature, 4500 rpm, for 10 minutes. Discard the supernatant, add 5 ml of AK-2YT, and culture overnight at 30°C, 250 rpm. Calculate stock volume the next day. The output volume = 6*Ax*10x+1 pfu / ml (X: dilution factor, Ax: number of clones at X-fold dilution ratio.)

[0143] 8. Phage monoclonal antibody screening

[0144] In a 96-well medium plate, add 400μl / well 2YT-AG (final concentration 50μg / mlAmp+, 2% glucose. Pick a single clone in a deep-well plate and mark it as PC (positive control) and NC (negative control). Remove the pipette tip and cover with sealing film. Culture overnight at 37℃ and 250rpm. Prepare 2YT-0.1% AG liquid culture medium, dispense 100μl / well into a 96-well deep-well plate, and add 5μl of the picked overnight single clone bacterial solution. Activate and culture at 37℃ and 250rpm for 2-3h (OD value is about 2). Prepare a 2YT+helper phage (MOI=5) mixture and add 20μl / well to the deep-well plate. Incubate at 37℃ for 30 minutes. Culture at 37℃ and 220rpm for 1.5h, then add 300μl 2YT-AK (final concentration 100μg / ml Amp+, 50 μg / ml Kan+) were added and incubated overnight at 30°C, 250 rpm, for 16-18 hours. The next day, the cells were centrifuged at 4000 rpm for 15 minutes at 4°C, and the supernatant was collected for ELISA analysis.

[0145] 9. Phage monoclonal ELISA test: (indirect method, competitive method)

[0146] Prepare a chemiluminescent plate and dilute 3-MT-DSS-BSA to 0.25 μg / mL in CB. Coat the plate overnight at 4°C at 100 μl / well. The next day, wash the plate twice with PBST, pat dry, and then add 150 μl / well of casein using a dispenser. Vortex the plate on a microplate oscillator to mix thoroughly. Block the plate at room temperature for 2 hours. Drain the liquid from the wells and pat dry with absorbent paper.

[0147] Indirect reaction: Add 50 μl / well of the phage centrifugation supernatant to the antigen-coated plate. Vortex and mix thoroughly on a microplate oscillator. Incubate at 37°C for 0.5 h. Wash with PBST five times. Pat dry. Add 100 μl / well of the enzyme-conjugate anti-M13-HRP (1 / 5k). Incubate at 37°C for 30 min. Wash the plate manually with PBST five times. Pat dry. Add 100 μl / well of luminescent substrate A and substrate B and mix thoroughly. Detect using a LUMO luminometer. Detect immediately and after 5 min. The 5-min result is the final result.

[0148] Competition reaction—3MT monoclonal competition screening strategy:

[0149] (1) Preliminary screening of superior clones: using only 2-point evaluation (very low, very high), using 0 pg / ml value and 1500 pg / ml calibrator;

[0150] (2) Simultaneous screening of main cross-reacting substances: 2-point detection of main cross-reacting substances NMN (low point, high point), 150 and 15000 pg / ml detection, screening of cross-reactivity low clones, retesting of superior clones: selection of calibration standards and clones with good cross-reactivity, retesting, increase of calibration standard gradient: 0, 40, 200, 1000 pg / ml, selection of 0 / 40-point superior clones;

[0151] (3) Increase of cross-reacting substance screening: NMN / MN repeated determination to determine normal cross-reactivity.

[0152] According to the indirect method of luminescence value, the bacteriophage is diluted to a luminescence value near 5w using diluent, 50ul / well of different calibration standards, preliminary screening of superior clones: only 2-point evaluation (extremely low, extremely high) is used, 0 pg / ml value and 1500 pg / ml calibration standards are used; simultaneous screening of main cross-reacting substances: 2-point detection of main cross-reacting substances NMN (low point, high point), 150 and 15000 pg / ml detection, 50ul / well of diluted bacteriophage is added to the antigen-coated plate. Shake and mix on a micro-vibrator, react at 37°C for 0.5h. Wash the plate manually, PBST washing solution, 5 times. Dry. Add enzyme conjugate anti-M13-HRP (1 / 5k), 100ul / well. React at 37°C for 30min. Wash the plate: wash the plate manually, PBST washing solution, 5 times. Dry. Color development: add luminescence substrate A+substrate B and mix, 100ul / well. Detect using a LUMO luminescence instrument. Detect immediately and after 5min. Take the 5min result as the standard. Selection of calibration standards and clones with good cross-reactivity, retesting, increase of calibration standard gradient: 0, 40, 200, 1000 pg / ml, selection of 0 / 40-point superior clones; increase of cross-reacting substance screening: NMN / MN repeated determination to determine normal cross-reactivity, the same as above.

[0153] Antibody sequencing: 20 superior bacteriophage strains are selected for sequencing.

[0154] 10. Full-length antibody construction

[0155] According to the sequencing results, different primers of rabbit monoclonal antibody variable regions are selected. Primer pairing scheme and optimal annealing temperature, PCR setting: PCR reaction system (25ul system): template DNA: 2.5ul F (10uM): 0.75ul R (10uM): 0.75ul 2x primerstar Max: 12.5ul high-pressure purified water: 8.5ul

[0156] PCR reaction parameter setting:

[0157] 98°C 5min;

[0158] 95°C 15s

[0159] 56℃ 15s 30cycles

[0160] 72℃ 25s

[0161] 72℃ 10min.

[0162] Nucleic acid gel recovery and purification.

[0163] 11. Homologous Recombination Reaction

[0164] Homologous recombination reaction system (10 μl): Vector: 50 ng, Fragment: 40 ng, 2× Homologous Recombinase: 5 μl, High-Prescription Water: Make up to 10 μl. Incubate at 50°C for 1 hour and then at 4°C. Transform into Top10 E. coli. Spread on a plate and incubate inverted at 37°C for at least 12 hours.

[0165] 12. Recombinant Screening

[0166] Pick a single colony and place it in 3 ml of LB liquid medium (Amp+), and culture it at 37°C with shaking at 220 rpm for 3 h.

[0167] Prepare PCR reaction solution (50 μl system)

[0168] F: 1.5 μl

[0169] R: 1.5 μl

[0170] 2×Taq MixPLUS: 25 μl

[0171] High-pressure purified water: 20 μl

[0172] Aliquot 15 μl into each tube and add 1 μl of bacterial solution

[0173] The PCR reaction parameters were set as follows:

[0174] 98℃ 5min

[0175] 95℃ 15s

[0176] 56℃ 15s 30cycles

[0177] 72℃ 25s

[0178] 72℃ 10min

[0179] Agarose gel electrophoresis was performed, positive clones were sequenced, and plasmids were extracted from the correctly sequenced clones.

[0180] 13.3-MT 4354# IgG Antibody Expression

[0181] For plasmid extraction, use the MN kit to extract the plasmid (mid-extraction);

[0182] Plasmid transient transfection

[0183] On the third day of 293F cell culture, sterile samples were taken for cell counting, and the cell density reached (3.0-5.0)×10 6 cells / ml, with a viability of ≥90%, discard an appropriate amount of cell suspension and add fresh culture medium to 500 ml, and adjust the cell density to 2.0×10 6 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and 130 rpm (amplitude 25 mm).

[0184] On the second day of cell culture, sterile samples were taken for cell counting, and the cell density reached (3.0-5.0)×10 6 cells / ml, with a viability of ≥90%, discard an appropriate amount of cell suspension and add fresh culture medium to 500 ml, and adjust the cell density to 3.0×10 6 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and 130 rpm (amplitude 25 mm).

[0185] To prepare a single 200mL cell suspension for transfection: In a clean bench, prepare two sterile 50mL centrifuge tubes, labeled A and B. Add the calculated light and heavy chain plasmids to tube A and gently shake to mix. Add 2mg / mL PEI to tube B and gently shake to mix. Incubate solution A and solution B separately at room temperature for 5 minutes. Then, pipette solution B into solution A to create a mixture of solution AB (see Table 1). Gently shake to mix, and incubate at room temperature for 10 minutes. After incubation at room temperature, pipette the solution AB slowly into a 1L shake flask, gently shaking to mix thoroughly. Incubate the tube in a shaker at 37°C, 5% CO2, and 130 rpm (25 mm amplitude).

[0186] Table 1

[0187]

[0188] 14.3-MT4354# Antibody Purification (SPA Method)

[0189] The purified supernatant sample, such as 250 ml, is passed through a 400 mesh nylon net, and the IgG concentration is determined by A280 (0.02M PBS pH 7.4 as blank buffer). The culture supernatant is filtered through a 0.45 μm filter membrane, and then is loaded for use. The filtered sample is diluted to 2 mg / ml with 0.02M PBS pH 7.4. The AKTA purification equipment is used for the experiment, and the SPA column is equilibrated, loaded, rinsed and eluted. After elution, the antibody is dialyzed in pbs buffer, concentrated by ultrafiltration tube, filtered by 0.22 μm filter membrane, and diluted to an antibody final concentration of 5.0±0.2 mg / ml with 0.01M PBS pH 7.2. The electrophoresis result is shown in Figure 1 The HPLC result is shown in Figure 2

[0190] The HPLC shows that the purity of 3-MT is >98%.

[0191] 15. Biotinylated antibody labeling of 3-MT4354#

[0192] According to Biotin / antibody=2:1 (molar ratio), 270 μl of Biotin is added into the 3-MT4354# antibody solution, and is shaken while dropping, and after dropping, it is fully mixed by vortexing for 16 hours at 25 degrees.

[0193] Example 2 Performance evaluation of the kit of the present application

[0194] The 3-MT4354# antibody labeled by biotin in Example 1 is used to coat magnetic beads, and is combined with HRP labeled 3-MT antigen and other components to prepare a magnetic microparticle 3-methoxy tyramine detection kit (magnetic microparticle chemiluminescence method). The detection is performed by using the competitive principle: the antibody coats the magnetic microparticles, the horseradish peroxidase labeled antigen, and the antibody-enzyme labeled antigen complex is formed by immune reaction, which catalyzes the light emitting substrate solution to emit photons, and the light intensity is inversely proportional to the content of the measured substance.

[0195] The 1605# and 4354# selected in Example 1 are respectively used as coating antibodies, and the plasma with liquid quality value is used as a sample to compare the performance difference between the two.

[0196] 1. Titer and gradient evaluation

[0197] Table 2

[0198]

[0199] The results show that the titer and gradient of the 4354# rabbit monoclonal antibody are better than those of the control antibody 1605#, which meets the needs of the development of the kit.

[0200] 2. Reproducibility

[0201] Table 3​

[0202]

[0203] The results showed that when the 4354# antibody was coated at 0.003 μg / T, the Q1 concentration value variation was 6.24%, the Q2 concentration value variation was 6.53%, and the Q3 concentration value variation was 5.60%. The repeatability could meet the requirements of the 3-MT project.

[0204] 3. Stability

[0205] Table 4

[0206]

[0207] Results: After 7 days of accelerated testing at 37℃, the signal value of 4354# calibration matrix and plasma matrix decreased by about -2.37%, and the concentration value variation was less than 10%. The stability can meet the requirements of project 3-MT.

[0208] 4. Crossover rate.

[0209] Table 5

[0210]

[0211] Note: Norepinephrine (NE), epinephrine (E), and dopamine (D) are catechol analogs.

[0212] Conclusion: The crossover rate with low values ​​of MN, NMN, NE, D, and E is <5%, and the crossover rate with high values ​​of MN, NMN, NE, D, and E is <15%, which meets the requirements for project 3-MT detection.

[0213] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. 3-methoxydopamine hapten, characterized in that Its structural formula is shown in Formula II: Formula II. 2.3-methoxydopamine complete antigen, characterized in that It is a conjugate of the 3-methoxydopamine hapten and a carrier protein according to claim 1; The carrier protein includes one or more of KLH, bovine serum albumin, human serum albumin, and ovalbumin; The carboxyl group at the terminal of the 3-methoxydopamine hapten is coupled to the amino group of the carrier protein.

3. The 3-methoxydopamine complete antigen according to claim 2, characterized in that Its structure is shown in Formula III: Formula III.

4. The method for preparing the complete 3-methoxydopamine antigen according to claim 2 or 3, characterized in that: The steps include: Synthesize a hapten with the structure of formula II; The hapten is coupled to the carrier protein, and the coupled product is dialyzed to obtain the complete 3-methoxydopamine antigen.

5. The preparation method according to claim 4, characterized in that The coupling reaction conditions are: shaking reaction at 18-27° C. overnight, and the shaking reaction time is ≥16 h.

6. Use of the hapten according to claim 1 or the complete antigen according to any one of claims 2 to 3 in any of the following: (1) Preparation of 3-MT recombinant monoclonal antibody; (2) Preparation of products for detecting the levels of 3-MT and its derivatives; (3) Preparation of diagnostic products for pheochromocytoma and / or paraganglioma. 7.3-MT recombinant monoclonal antibody, characterized in that This includes any of the following: (1) A 3-MT recombinant monoclonal antibody produced by immunizing an animal with the complete 3-methoxydopamine antigen according to claim 2 or 3; (2) 3-MT recombinant monoclonal antibody, the three CDR regions of its light chain have: A), the amino acid sequence shown in SEQ ID NO: 1 to 3; or B) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in A); or C) has at least 80% of the sequence shown in A) or B); The three CDR regions of its heavy chain have: D), the amino acid sequence shown in SEQ ID NO: 4 to 6; or E), a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in D); or F) and the sequence shown in D) or E) have at least 80% of the sequence.

8. The 3-MT recombinant monoclonal antibody according to claim 7, characterized in that It includes a heavy chain variable region and a light chain variable region; The light chain variable region has: G), the amino acid sequence shown in SEQ ID NO: 7; or H) a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in G); or I) and the sequence shown in H) or I) have at least 80% of the sequence; The heavy chain variable region has: J), the amino acid sequence shown in SEQ ID NO: 8; or K), a sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in J); or L), and the sequence shown in J) or K) have at least 80% of the sequence.

9. Biomaterial, characterized in that Includes any of the following: (I) a nucleic acid encoding the 3-MT recombinant monoclonal antibody according to any one of claims 7 to 8; (II), an expression cassette or recombinant vector comprising the nucleic acid described in (I); (III) A host comprising the nucleic acid of (I) or the recombinant vector of (II).

10. Use of any one of the following a) to b) in the preparation of a detection product: a), the 3-MT recombinant monoclonal antibody according to any one of claims 7 to 8; b) the biomaterial according to claim 9; The test products include: Products for detecting the level of 3-MT or its derivatives, or diagnostic products for pheochromocytoma and / or paraganglioma.

11. A kit comprising the complete antigen according to claim 2 or 3 and / or the 3-MT recombinant monoclonal antibody according to any one of claims 7 to 8.

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